1. Field of the Invention
The present invention relates to a method of separating a protein concentrate from foliage of leafy green plants. These concentrates generally are referred to as leaf protein concentrates or LPC. More particularly, the present invention relates to a method of precipitating soluble protein fractions and simultaneously coagulating suspended protein fractions from green juice expressed from macerated leaf tissue, without the requirement for high temperature treatments, i.e., steam, or for the utilization of dangerous or costly chemical precipitants.
2. Description of the Prior Art
The desirability of separating protein from a very inexpensive protein source, such as forage crops, has been recognized for many years. Laboratory efforts directed toward this end have been carried on since at least 1940 for the purpose of finding a commercially viable method of extracting plant protein as a nutritional supplement for humans, and as an animal feed. The removal of a substantial portion of the protein from tobacco has been examined for the purpose of producing a more pleasant smelling tobacco smoke and a safer product with the additional benefit of providing a useful protein by-product for human consumption, as set forth in the article, "Recent Advances In Chemical Composition in Tobacco and Tobacco Smoke", by DeJong et al, Proceedings of American Chemical Society Symposium, 1977.
Proteins have been precipitated from solution, particularly in the laboratory, by many techniques. Soluble protein has been precipitated by adding precipitants to solution, for example, the addition of polar solvents, mineral salts and polyelectrolytes. One common technique for precipitating protein from solution is by adjusting the pH of the protein solution to the protein isoelectric point with the addition of an organic or mineral acid. Isoelectric precipitation of soluble protein has been made commercial, but it has been found that the protein precipitate is very difficult to remove from the solution since the precipitate has a very fragile particulate structure and therefore it is difficult to coagulate and sediment.
Another very common method of precipitating and coagulating soluble protein from solution is by heating the protein solution, the heat being supplied usually in the form of steam. A substantial amount of energy is required to perform the heat precipitation method and very serious problems can result from the heat precipitation technique. The heat precipitated protein product can suffer irreversible damage in heating, thereby lowering the nutritional value of the product. Further, the heat precipitation technique, although commercially feasible, is associated with processing problems because high temperature protein precipitation produces a protein product which is very adhesive in nature so that the protein product continually fouls interior surfaces of pumps, hoses, tanks and other transport and storage apparatus.
Glutaraldehyde has been reacted with protein to form water-insoluble, enzymatically active products for specialized use in column or filtration techniques, to remove or alter substances (see Jansen and Olson, Archives of Biochemistry and Biophysics, volume 129, No. 1, January, 1969, pages 221-227), and for fixation of the protein for study (see Science, Mar. 31, 1967, Vol. 155, No. 3770, pages 1672-1674).
One of the problems experienced during the reaction of glutaraldehyde with protein is that the reaction proceeds very slowly, particularly at low pH levels. As set forth by Jansen and Olson (Table 1, page 223, Archives of Biochemistry and Biophysics), a time period of about 24 hours is required for the occurrence of the first visual precipitate when protein containing .epsilon.-amino groups (i.e., papain) is reacted with glutaraldehyde at a pH of about 5.0 or lower.
The Leroy et al. U.S. Pat. Nos. 4,211,795 and 3,507,662 disclose a process for reacting an animal feed with a tanning substance (i.e., glutaraldehyde) to form a more insoluble feed to substantially decrease the deaminization of proteins in the ruminant of animals. The Leroy et al. patents, however, do not teach or suggest the reaction of glutaraldehyde with protein in solution for the purpose of precipitation and, particularly, do not teach the use of glutaraldehyde for precipitation of proteins from green juice. Further, because of the condition of the animal feed, when reacted, a substantial amount of contact time is required to permit the feed to absorb the glutaraldehyde for reaction.
The Kalb et al. U.S. Pat. No. 3,823,129 discloses a process of reacting a particular protein (Concanavalin A--a protein contained in Jack beans) with glutaraldehyde to insolubilize the protein so that the insolubilized protein can be repeatedly used for the recovery of transition metals and saccharides.
The Batley et al. U.S. Pat. No. 4,130,553 discloses a method of precipitating protein from green juice by raising the pH to a level of at least 9.0 and discloses, in the background of the invention, a common prior art heat precipitation technique wherein the green juice is heated to a temperature of at least 70.degree. C. and the pH of the juice is adjusted to a value below 5.0.
The Bickoff et al. patent 3,823,128 discloses a method of precipitating protein from green juice by adding a source of sulfite ions, such as sodium sulfite or bisulfite or potassium sulfite or bisulfite. In accordance with the Bickoff et al. invention, the precipitated material has a jelly-like consistency and is heated to a temperature of 70.degree.-90.degree. C. to change the jelly-like material into a curd material, at an alkaline pH, so that the precipitate can be removed.
The Amotz et al. U.S. Pat. No. 4,116,771 discloses a method of insolubilizing enzymes by a crosslinking reaction with glutaraldehyde so that the resulting enzyme product may be used in such environments as an enzyme reactor and the like.
Two published government owned patent applications Ser. No. 897,083 filed Apr. 17, 1978 and Ser. No. 15,491, filed Feb. 26, 1979 disclose, similar to the above described Leroy et al. U.S. Pat. Nos. 4,211,795 and 3,507,662, contacting an animal feed with a particular reactant to protect the animal feed from ruminal degradation. Neither of these published government applications suggest that glutaraldehyde can react with protein at a particularly low pH to cause easy and fast precipitation of protein from green juice resulting in a curd coagulant easily removable from processing equipment.
Other uses for glutaraldehyde have been disclosed such as in U.S. Pat. No. 2,941,859 (leather tanning); U.S. Pat. No. 3,342,543 (wool stabilization); and U.S. Pat. No. 3,294,564 (protein alteration). Prior to the present invention, however, the crosslinking reagents disclosed herein have not been used to precipitate and coagulate protein from green juice at a pH in the range of 3.0-5.0. The glutaraldehyde reaction carried out in leather tanning and wool stabilization proceeds over a wide range of pH, although an alkaline pH generally is preferred. The crosslinking reaction of the present invention is a substantially different reaction than the glutaraldehyde reaction in wool stabilization or leather tanning as evidenced by the fact that the reaction disclosed herein cannot be carried out at an alkaline pH.
In accordance with an important feature of the present invention, the protein curd separated from green juice has little to no phenolic compounds covalently bound to the separated protein. Apparently, this phenomenon results from the fact that the .epsilon.-amino groups of the lysines of the protein separated in accordance with the present invention are tied to one another by virtue of the crosslinking reagent as defined herein, rather than being free as a reaction site for polyphenols or other deleterious reactants. It is well known that polyphenols often cause nutritional difficiencies and toxicities when proteins containing substantial amounts of polyphenols are used for animal feeds, as shown for protein extracted from high-tannin species of sorghum (see Whitaker et al. Chemical Deteri-oration of Proteins, ACS Symposium Series 123, 1980 Chapter 9 pages 202-206 and Chapter 1, pages 1-14). Because amino groups are excellent and because there are invariably many epsilon amino groups of lysines on the surface of proteins, it is an important feature of the present invention that the separated protein has substantially all of the lysine amino groups tied together, in the same or adjacent molecules, so that they are not available as reaction sites. Accordingly, the protein separated in the manner prescribed by the present invention is not subject to the common deteriorative reactions involving lysine.